We have further characterized various cell types in the mouse thymus by biochemical phenotypic, functional and/or differentiation potential. We have shown that T cell receptor (TCR) gamma delta + CD-8- (double negative) thymocytes are the major source of such cells in the peripheral lymphoid organs. A minor extrathymic pathway also exists for generating gamma delta + T cells. The TCR gamma delta on thymocytes is associated with the same CD3 components and is activated by the same stimuli that activates TCR alpha beta T cells. A TCR alpha beta + double negative the thymocyte was also identified. These cell have many of the characteristics of mature T cells but are distinguished by a restricted TCR V beta repertoire. Intrathymic transfer of isolated subsets of thymocytes into irradiated recipients revealed that the TCR alpha beta+ cells can be derived from CD4+ 8- thymocytes. In strains of mice in which TCR V beta 8.1-bearing thymocytes are deleted as a means of removing potentially self-reactive T cell clones, the TCR alpha beta+ double negative thymocytes show an enrichment for cells using the V beta 8.1 receptor. Finally, obtained with in vivo anti-CD4 antibody treatment support the hypothesis that CD4+8+ thymocytes are the precursors of single positive thymocytes and that self-tolerance by clonal deletion occurs at the CD4+8+stage of development.